Microchannel gel electrophoretic separation systems and methods for preparing and using

摘要

A micro-analytical platform for performing electrophoresis-based immunoassays was developed by integrating photopolymerized cross-linked polyacrylamide gels within a microfluidic device. The microfluidic immunoassays are performed by gel electrophoretic separation and quantifying analyte concentration based upon conventional polyacrylamide gel electrophoresis (PAGE). To retain biological activity of proteins and maintain intact immune complexes, native PAGE conditions were employed. Both direct (non-competitive) and competitive immunoassay formats are demonstrated in microchips for detecting toxins and biomarkers (cytokines, c-reactive protein) in bodily fluids (serum, saliva, oral fluids). Further, a description of gradient gels fabrication is included, in an effort to describe methods we have developed for further optimization of on-chip PAGE immunoassays. The described chip-based PAGE immunoassay method enables immunoassays that are fast (minutes) and require very small amounts of sample (less than a few microliters). Use of microfabricated chips as a platform enables integration, parallel assays, automation and development of portable devices.

主权项

1. A method for preparing a separation system for electrophoretically separating chemical and/or biological species, comprising:
providing a capillary separation channel disposed on a substrate, comprising:
a fluid reservoir disposed at each of first and second ends of the capillary separation channel, wherein the fluid reservoirs and the capillary separation channel filled with a first solution mixture comprising an acrylamide monomer, a bis-acrylamide monomer, and a UV photoinitiator having a first predetermined concentration of total acrylamide;a mixing and incubation region proximal to the first end of the capillary separation channel; anda plurality of adjacent side channels each having at least one fluid reservoir in fluid communication with the side channel, wherein each of the at least one fluid reservoirs contains a quantity of one of a plurality of second solution mixtures, wherein each of the second solution mixtures again comprises an acrylamide monomer, a bis-acrylamide monomer, and a UV photoinitiator and wherein each of the second solution mixtures comprise a plurality of predetermined concentrations of total acrylamide which may be the same or different than the first predetermined concentration; introducing a measured portion of the second solution mixtures contained within two or more of the side channels reservoirs into the capillary separation channel until each of the solution mixtures contacts an adjacent solution mixture along a contact interface; mixing each of the plurality of first and second solution mixtures within the capillary separation channel by diffusion for a period of time; simultaneously exposing the mixed solution mixtures within the capillary separation channel to a quantity of UV light polymerizing the mixed solution mixture and thereby providing a length of polyacrylamide gel having a gradient in total acrylamide concentration; and providing a programmable high voltage power supply in electrical communication with the first and second ends of the capillary separation channel, the high voltage power supply providing an electric field within each of the capillary separation channels, wherein the electric field and the depth and width of the capillary separation channels cooperate to provide electrophoretic separation of a species complex introduced into the capillary separation channel in less than about 60 seconds.